Diabetes is a disease which effects many Americans. It is a disorder of carbohydrate metabolism characterized by hyperglycemia and glycosuria and resulting from inadequate production or utilization of insulin. The disease takes several different forms and is generally classified into two syndromes: Type I, or insulin-dependent diabetes mellitus (IDDM) and type II, or non-insulin-dependent diabetes mellitus (NIDDM). In Type I the patients secrete little or no insulin. In Type II the individual is able to produce insulin but for whatever reason the individual does not secrete enough insulin to control hyperglycemia. Type II is the most prevalent form of the disease and effects approximately 2-4% of individuals. Current treatment of diabetes Type II (NIDDM) involves constant maintenance of diet and often insulin therapy.
Ordinarily, after insulin is secreted by the xcex2 cells of the pancreas it is transported to the cells of the body where it binds to specific insulin receptors. The insulin then acts to promote glucose metabolism in the cell and to inhibit glucose production by the liver.
The basic cause for diabetes is still uncertain but the pathological defect involves failure of the xcex2 cells of the pancreas to secrete an adequate amount of insulin. In the absence of sufficient insulin, gluconeogenesis is stimulated, glycolysis is adversely affected and glucose uptake is reduced. All of these factors contribute to diabetic hyperglycemia.
Glucose is the primary regulator of insulin secretion. It is well established that glucose must be metabolized for insulin secretion to ensue. The rate of insulin secretion closely parallels its metabolic rate and inhibitors of glucose metabolism effectively block insulin secretion. In all cells glucose metabolism is initiated by hexokinase-catalyzed phosphorylation. Unique to the pancreatic xcex2 cell and the hepatocyte of the liver, this reaction is catalyzed by an unusual hexokinase isoform, hexokinase IV (commonly referred to as glucokinase due to its relative specificity for glucose as a substrate). Hexokinase IV has a Km for glucose in the physiological range of glucose levels, ≈10 mM. This is almost two orders of magnitude higher than any other mammalian hexokinase. Therefore, in vivo hexokinase IV-catalyzed phosphorylation is proportional to blood glucose levels, whereas phosphorylation by other hexokinases is saturated at physiological glucose levels. Hexokinase IV is also distinct from other hexokinases in that it is not allosterically inhibited by its product, glucose 6-phosphate (G6P). Due to the high Km of hexokinase IV and the absence of end-product inhibition, glucose phosphorylation in the xcex2 cell closely parallels circulating glucose concentrations. It has therefore been proposed that hexokinase IV functions as the glucose receptor or glucose sensor of the pancreatic xcex2 cell.
In non-insulin-dependent diabetes mellitus (NIDDM), there is a failure to secrete adequate amounts of insulin despite the fact that pancreatic islets retain significant levels of insulin. (Stefan, Y. et al. (1982) Diabetes 31,694-700.) The secretory deficit appears to be specific for glucose-induced secretion as other secretagogues retain normal efficacy. (Palmer, J. P. et al, (1976) J. Clin. Invest. 58,565-570). The hypothesis that hexokinase IV may serve as the xcex2-cell glucose sensor has taken on great clinical significance recently since it has been demonstrated that a nonsense mutation in only one allele of the hexokinase IV gene predisposes to some types of NIDDM. (Vionnet, N. et al (1992) Nature (London) 356,721-722.) As hexokinase IV is a monomeric enzyme, these results suggest that a simple 50% reduction in hexokinase IV activity due to mutational inactivation of one allele may lead to diabetes.
Thus it can be seen a need exists in the art for further study of the causes of diabetes which may lead to a potential cure, as well as a test of the hypothesis that hexokinase IV may serve as the glucose xcex2-cell sensor. It is an object of the present invention to provide a transgene which expresses a 2-fold increase in hexokinase activity for study of the role of hexokinase in insulin secretion and diabetes.
It is another object of the present invention to provide a method of decreasing diabetes by stimulating hexokinase activity and thereby increase insulin secretion for Type II diabetic patients.
It is yet another object of the present invention to provide an animal model for study of diabetes or other glucose regulatory mechanisms in which the animal model experiences a 2-fold increase in hexokinase activity which is specific to the xcex2 cell.
The present invention relates to construction of a transgene comprising the yeast hexokinase B gene (yeast hexokinase B like hexokinase IV is not allosterically inhibited by intracellular levels of G6P) operatively linked to an insulin promoter. The transgene, when inserted into mice resulted in a 2-fold increase in hexokinase activity. The increase was found to significantly augment glucose stimulated insulin secretion of isolated pancreatic islets, to increase serum insulin levels in vivo, and to lower blood glucose levels of transgenic animals by 20-50% below control levels.
The transgene of the present invention may be introduced into an animal model for study of the disease, or may be used for direct manipulation of pancreatic xcex2 cells for possible treatment for diabetes.